Agaricus bisporus (button mushroom) strain, mushroom compost, and casing soil
Commercial white (192915 AG, Soc, France) strain of A. bisporus, mushroom compost and casing soil were obtained from SMS Ersanlar company in Korkuteli county, Antalya province, Turkey. Trademarks of compost and casing soil were Ersan and Çivril, respectively. Composition of the casing soil consisted of organic matter, silt, and various elements (e.g. C, N, P, K, Ca, Mg). No treatment was applied to the casing soil.
Green mold isolates
Two isolates, Çom1 (Accession number: MH133213 in Genbank (http://www.ncbi.nlm.nih.gov)) and K39 (Accession number: MH133214) of T. aggressivum f. aggressivum, were used from culture collection of the mycology laboratory of Batı Akdeniz Agricultural Research Institute. Both isolates, Çom1 and K39, were previously isolated from mushroom compost in Çomaklı village and Akyar district of Antalya province, respectively in 2015 (Aydoğdu et al. 2020). The isolates were grown on potato dextrose agar (PDA) at 25 °C for 10 days. Under these conditions, they initially formed whitish colonies, and 4 to 5 days later, colonies began to turn green as a result of sporulation.
Bacterial isolates
In 2019, five bacteria were isolated from mushroom compost (Phase 3). Samples were obtained from mushroom farms in the Korkuteli county, Antalya province. These bacterial isolates were designated as MSG-5, MSG-11, MSG-15, SGM-1, and SGM-2.
Identification of the bacterial isolates
Morphological and biochemical traits of each bacterial isolate were examined in vitro. Gram reactions of the bacterial isolates were determined by mixing bacteria with a drop in 3% solution of KOH (Suslow et al. 1982). Biochemical traits of the bacterial isolates were characterized according to the standard methods of Bergey’s Manual of Determinative Bacteriology (Holt et al. 1994).
To confirm morphological and biochemical identification, molecular diagnosis was also performed. DNA of each bacterium was extracted and purified, using Promega DNA purification kit. 16S rRNA was amplified, using universal oligonucleotide primers, 27F (5′-AGA GTT TGA TCC TGG CTC AG-3′) and 1492R (5′-GGT TAC CTT GTT ACG ACT T-3′) (Lane 1991; Kadaikunnan et al. 2015). In addition, gyrA gene was amplified, using primers 5′-CAG TCA GGA AAT GCG TACGTC CTT-3′ (forward primer) and 5′-CAA GGT AAT GCT CCA GGC ATT GCT-3′ (reverse primer) (Chun and Bae 2000).
PCR products were seperated from 1.5% agarose gels, stained with safe DNA dye and visualized under UV light. Sequence analysis was done by Letgen (Kazım Dirik Mah. 296/2 Sok. No:33 35100 Bornova/İzmir-Turkey). The sequences were compared to BLAST searches (http://www.ncbi.nlm.nih.gov) to find the closest sequence matches and deposited at the GenBank.
In vitro experiments
Dual culture tests were used to examine effects of the bacterial isolates on mycelial growth of T. aggressivum f. aggressivum and A. bisporus. Owing to weak mycelial growth of A. bisporus on artificial media (e.g., potato dextrose agar and so on), compost extract medium (CEM) was prepared as described by Rainey (1989) and used to secure mycelial growth of A. bisporus in vitro experiments.
Effects of bacterial isolates on T. aggressivum f. aggressivum and A. bisporus
One mycelial plug (0.5 cm) of each isolate of T. aggressivum f. aggressivum was placed at nearly 2-cm away from the edge of a Petri dish (9 cm in diameter) containing CEM and a loopful of each bacterial isolate was streaked to the opposite side. The same process was performed for each interaction and incubated at 25 °C for 7 days. The experiments were set up according to the completely randomized factorial design with four replications. In the controls, only mycelial plugs of the isolates of T. aggressivum f. aggressivum were placed. In each treatment, colony radial growth of T. aggressivum f. aggressivum was measured, using a caliper and compared with the control. Five days later, growth inhibition of T. aggressivum f. aggressivum caused by each bacterial isolate was determined, using the formula below (Vinale et al. 2008).
$$ \mathrm{Growth}\ \mathrm{inhibition}\ \mathrm{ratio}\ \left(\%\right)=\left(\mathrm{R}1-\mathrm{R}2\div \mathrm{R}1\right)\times 100, $$
where R1 = colony radial growth (mm) of T. aggressivum f. aggressivum in the control and R2 = colony radial growth (mm) of T. aggressivum f. aggressivum in the treatment.
Three spawn grains covered by white strain (192915 AG, Soc, France) of A. bisporus were placed on CEM and incubated at 25 °C for 7 days. To use in dual culture tests, mycelial plugs (0.5 cm) of the strain of A. bisporus were taken from the 7-day-old colonies growing on CEM, and as aforementioned, dual culture tests were conducted with the bacterial isolates. The experiments were set up according to the completely randomized design with 4 replications. In the controls, only mycelial plugs of the strain of A. bisporus were placed on CEM. In each treatment, colony radial growth of the strain of A. bisporus was measured, using a caliper and compared with the control. Fourteen days later, growth inhibition of A. bisporus caused by each bacterial isolate was determined, using the above formula of Vinale et al. (2008).
In vivo experiments
Commercial biological preparations
In addition to the native bacterial isolates, 2 commercial bacterial preparations and one fungal biological preparation were also used in vivo experiments. These commercial preparates were designated as Tic-1, Tic-2, and Tic-3, and their commercial names were Serenade (SC), Cedriks (SL), and Ovnier (SL), respectively.
Preparation of bacterial suspensions of the native isolates
Bacterial suspension of each isolate was prepared from 2-day-old cultures grown on nutrient agar medium. Inoculum concentration was adjusted to 108 cfu/ml, using sterile distilled water in a spectrophotometer (SHIMADZU UV-120-01).
Preparation of green mold inoculum
Each isolate of T. aggressivum f. aggressivum was incubated on PDA at 25 °C for 10 days. Later, 12 ml sterile distilled water per Petri dish was added to the colony of each isolate growing on PDA. Spore suspension was sieved through a muslin and transferred to a 2-l beaker. Spore density was adjusted to 1 × 106 conidia/ml, using a hemocytometer.
Preparation of casing soil
Commercial casing soil was passed through a sieve to remove big pieces from it.
Providing mushroom-growing conditions
Mushroom-growing conditions (temperature, humidification, and ventilation arrangements) were provided in a room in the basement of the Department of Plant Health of Batı Akdeniz Agricultural Research Institute, Antalya province. For assessments of biological control of green mold in compost and in casing soil, 2 separate experiments were conducted.
Compost and casing soil experiments
Compost experiment was carried out according to a completely randomized design in factorial with 3 replications. Each experimental unit consisted of one bag (40 × 25 cm) containing 2.2 kg compost and 660 g casing soil. Initially, 2.2 kg compost spawned with white strain (192915 AG, Soc, France) of A. bisporus was added into each bag and incubated at 25 °C for 7 days. Within this period, the strain of A. bisporus partially colonized the compost. Afterwards, 3 ml inoculum (1 × 106 conidia/ml) of each isolate of T. aggressivum f. aggressivum was injected into the midsection of each bag using a syringe. Three days later, in the treatments, 5 ml suspension (108 cfu/ml) of each bacterial isolate was injected into the compost nearly 5 cm above from the section in which T. aggressivum f. aggressivum inoculum was given. Commercial biological preparations were applied as the same way for each bag in the treatment plots (A. bisporus + T. aggressivum f. aggressivum + Bacterial isolate/Commercial biological preparation).
In the experiment, two separate controls were arranged for each treatment plot. In negative controls, neither T. aggressivum f. aggressivum nor the bacterial isolates/commercial biological preparates were applied to compost, but only the strain of A. bisporus grew in the compost. In positive controls, no bacterial isolates/commercial biological preparations were applied but just green mold inoculum (T. aggressivum f. aggressivum) was given into the compost containing the strain of A. bisporus. All bags were kept at 25 °C for 10 days with 85 to 90% relative humidity. Afterwards, casing soil (660 g) was added to the top of each bag. Ten days later, temperature in the room was reduced 1 °C per day until reaching to 17 °C. Afterwards, ventilation providing oxygen from outside into the room was initiated and thus carbon dioxide level in the room was reduced. Casing soil was daily watered by spraying sterile water to keep its dampness. Ensuing emergence of fruiting bodies (sporophores) of the strain of A. bisporus, mushrooms in marketing size were picked and weighed per bag. Yield values of each treatment, negative and positive control plots, were calculated separately. Efficacy of each treatment was assessed by comparing both control plots.
Casing soil experiment was conducted according to a completely randomized factorial design with 3 replications. Each experimental unit was composed of one bag (2.2 kg compost + 660 g casing soil). Initially, neither T. aggressivum f. aggressivum nor the bacterial isolates were applied to the compost in this experiment. The compost containing spawn of the strain (192915 AG, Soc, France) of A. bisporus was kept at 25 °C for 19 days. Within this period, mycelium of the strain of A. bisporus colonized the compost in each bag. Later, casing soil (660 g) per bag was laid on the top of the compost in each bag. Addition of casing soil formed nearly 4- to 5-cm-thick layer on the top of the compost. In this stage, in the treatments, 3 ml inoculum (1 × 106 conidia/ml) of each isolate of T. aggressivum f. aggressivum was injected into central part of the casing soil. Following inoculation of T. aggressivum f. aggressivum, 1 day later, 5 ml bacterial suspension (108 cfu/ml) of each bacterial isolate was injected into the same central part of the casing soil in the treatments. The commercial biological preparations were aplied as the same way for each bag in the treatment plots. In the experiment, 2 separate controls were arranged as aforementioned in compost experiment. In addition, as aforementioned, the same mushroom-growing conditions were maintained, and the same experimental evaluations were done.
Statistical analysis
Analysis of variance (ANOVA) was done using SAS 9.1 software program (SAS Institute Inc., Cary, NC, USA). In vitro and in vivo experiments were conducted using 4 and 3 replicates for each treatment in each experiment, respectively. In vitro experimental data were analyzed using one- and two-way ANOVA, while in vivo experimental data were analyzed using three-way ANOVA. Mean values of each experiment in vitro and in vivo were grouped according to Tukey’s multiple range test with significance level of 1%. Dunnett test (P < 0.01) was also used to compare treatment plots with positive and negative control plots in in vivo experiments.
